Viral vaccines

ABSTRACT

A mutant virus for use as a vaccine for prophylaxis or therapy, wherein the genome of the virus is defective in respect of a gene essential for the production of infectious virus. In one aspect the mutant virus, e.g. a herpesvirus, e.g HSV-1 or HSV-2, is capable of protecting a susceptible species immunised therewith against infection by the corresponding wild-type virus. In another aspect, the mutant virus acts as a vector for an immunogenic protein derived from a pathogen, encoded by foreign DNA incorporated in the mutant virus. The mutant virus can be produced by a recombinant host cell which expresses a gene complementing the defect. The mutant virus can be infectious for the host to be protected, and the genetic defect can allow expression in the infected host of at least some of the viral genes, which can provoke a cell-mediated immune response. The defect can be in a glycoprotein gene such as gH.

This application is a continuation-in-part of copending U.S. Ser. No.08/384,963 filed 7 Feb. 1995, which is itself a continuation of U.S.Ser. No. 08/030,073, corresponding to International patent applicationPCT/GB91/01632, which was published on 2 Apr. 1992 as WO 92/05263,having an international filing date of 23 Sep. 1991 and entered into USnational phase with serial number U.S. Ser. No. 08/030,073 and date 20May 1993.

This application is also a continuation-in-part of copending U.S. Ser.No. 08/216,260 filed 21 Mar. 1994, which is itself acontinuation-in-part of U.S. Ser. No. 08/168,643 filed 16 Dec. 1993. Thespecifications of the above-mentioned applications are herebyincorporated by reference.

The present invention relates to viral vaccines. In particular, itrelates to genetically engineered mutant viruses for use as vaccines; tovaccines comprising the mutant viruses; recombinant cells; and tomethods relating to the production of vaccines.

Viral vaccines are traditionally of two sorts. The first sort are‘killed’ vaccines, which are virus preparations which have been killedby treatment with a suitable chemical such as beta-propiolactone. Thesecond type are live ‘attenuated’ vaccines, which are viruses which havebeen rendered less pathogenic to the host, either by specific geneticmanipulation of the virus genome, or, more usually, by passage in sometype of tissue culture system. These two types of vaccine each havetheir own disadvantages.

Killed vaccines do not replicate in the host, and they must beadministered by injection, and hence may generate an inappropriate kindof immune response. For example the Salk vaccine, a killed preparationof poliovirus, produces an immunoglobulin (Ig) G antibody response, butdoes not stimulate the production of IgA in the gut, the natural site ofprimary infection. Hence this vaccine, though it can protect theindividual from the neurological complications of poliomyelitis, doesnot block primary infection, and so does not confer “herd immunity”.

In addition, killed viruses do not enter and replicate inside hostcells. Hence any beneficial immunological response to non-structuralproteins produced during replication is not available. They also cannotstimulate the production of cytotoxic T cells directed against virusantigens. “Dead” antigens can be picked up by antigen presenting cellsand presented to T cells. However, the presentation occurs via MHC ClassII molecules and leads to stimulation of T helper cells. In turn, the Thelper cells help B cells to produce specific antibody against theantigen. In order to stimulate the production of cytotoxic T cells,virus antigens must be processed through a particular pathway inside theinfected cell, and presented as broken-up peptide fragments on MHC ClassI molecules. This degradation pathway is thought to work mosteffectively for proteins that are synthesised inside the infected cell,and hence the only virus that enters host cells and expressesimmunogenic viral protein is capable of generating virus-specificcytotoxic T cells. Therefore, killed vaccines are poor inducers ofcellular immunity (cytotoxic T cells) against virus infection. From thispoint of view, live attenuated vaccines are more satisfactory.

Live attenuated viruses have been made hitherto by deleting aninessential gene or partly damaging one or more essential genes (inwhich case, the damage is such that the genes are still functional, butdo not operate so effectively). However, live attenuated viruses oftenretain residual pathogenicity which can have a deleterious effect on thehost. In addition, unless the attenuation is caused by a specificdeletion, there remains the possibility of reversion to a more virulentform. Nevertheless, the fact that some viral protein production occursin the host means that they are often more effective than killedvaccines which cannot produce such viral protein.

Live attenuated viruses, as well as being used as vaccines in their ownright, can also be used as “vaccine vectors” for other genes, in otherwords carriers of genes from a second virus (or other pathogen) againstwhich protection is required. Typically, members of the pox virusfamily, e.g. vaccinia virus, are used as vaccine vectors. When a virusis used as a vaccine vector, it is important that it causes nopathogenic effects. In other words it may need to be attenuated in thesame way that a simple virus vaccine is attenuated. The samedisadvantages as those described above therefore apply in this case.

It has been found possible to delete a gene (especially, an essentialgene) from a viral genome and (also) provide a so-called “complementing”cell which provides the virus with the product of the deleted gene. Thishas been achieved for certain viruses, for example adenoviruses,herpesviruses and retroviruses. For adenoviruses, a human cell line wastransformed with fragments of adenovirus type 5 DNA (F L Graham, JSmiley, W C Russell and R Nairn, J Gen Virol, 36 (1977) 59-72). The cellline expressed certain viral genes, and it was found that it couldsupport the growth of virus mutants which had those genes deleted orinactivated (T Harrison, F Graham and J Williams, Virology 77 (1977),319-329). Although the virus grew well on this cell line (the“complementing cell line”) and produced standard viral particles, itcould not grow at all on normal human cells. Cells expressing theT-antigen-encoding region of the SV40 virus genome (a papovavirus) havealso been shown capable of supporting the replication of virusesspecifically deleted in this region (Y Gluzman, Cell, 23 (1981),182-195). For herpes simplex virus, cell lines expressing the gBglycoprotein (W Cai et al, J Virol 62 (1987), 714-721) the gDglycoprotein (M W Ligas and D C Johnson, J Virol 62 (1988) 1486-1494)and the Immediate Early protein ICP4 (N A Deluca et al, J Virol 56(1985) 558-570) have been produced, and these have been shown capable ofsupporting the replication of viruses with specifically inactivatedcopies of the corresponding genes.

According to the present invention, there is provided a mutant virus foruse as a vaccine, in which a viral gene encoding a protein which isessential for the production of infectious virus has been deleted orinactivated: and wherein said virus can be grown in a cell which has aheterologous nucleotide sequence which allows said cell to express theessential protein encoded by said deleted or inactivated viral gene.Such a mutant virus with a genome defective in respect of an essentialgene can protect a susceptible species immunised therewith againstinfection by e.g. the corresponding wild-type virus. As discussed below,the mutant virus in such a vaccine can be infectious for cells of asusceptible species, e.g. a mammalian species, immunised therewith.Viral protein can thereby be expressed in the cells.

The present invention also provides a vaccine which comprises a virus asdescribed above, together with one or more excipients and/or adjuvants.The viral genome may itself provide the immunogen. In certainembodiments it can contain genetic material such as a heterologous geneinsert expressing an immunogenic protein, e.g. from a pathogen exogenousto the virus. In such a case exogenous immunogenic protein can beexpressed in cells of a susceptible species immunised with the vaccinecontaining the mutant virus and infected by the mutant virus of thevaccine. Immunity against the pathogen can thereby be conferred in aspecies normally susceptible to the pathogen. The exogenous protein canbe from e.g. an immunodeficiency virus, and can be an immunodeficiencyvirus glycoprotein.

The mutant virus of the vaccine can be one that is capable in aninfected species of establishing a latent infection with periodicreactivation. The mutant virus can be such that the defect in theessential gene allows the mutant virus still to infect normal cells andreplicate therein to give rise to the production and release from thecells of non-infectious viral particles, but not to give rise toinfectious viral particles.

The present invention also provides a complementing cell transfectedwith an attenuated virus as described above, for use in the preparationof a vaccine.

The present invention also provides a method which comprises the use ofa virus as described above in the preparation of a vaccine for thetherapeutic or prophylactic treatment of a disease, and for prophylacticor therapeutic use in generating an immune response in a subjectinfected therewith.

The present invention also provides a method for the production of avaccine which comprises: culturing a cell infected with a virus having adeleted or inactivated viral gene encoding a protein which is essentialfor the production of infectious virus, and wherein the host cell has aheterologous nucleotide sequence comprising said viral gene and which isable to express the essential protein encoded by said gene; harvestingthe viral thus produced, and using it in a vaccine.

The mutant can be from a double-stranded DNA virus, e.g. a herpesvirus,e.g. a herpes simplex virus (HSV). The mutant can be a type-1 HSV or atype-2 HSV. The defect can be in for example the glycoprotein gH gene.

It can be seen that the invention provides a mutant non-retroviral viruswhose genome is defective in respect of a gene essential for theproduction of infectious virus, such that the virus can infect normalcells and undergo replication and expression of viral antigen genes inthose cells but cannot produce normal infectious virus.

The applicants have termed the mutant viruses described herein DISCviruses (standing for ‘defective infectious single cycle viruses’) andan outline of the concept is illustrated in FIG. 35 of the accompanyingdrawings. Such DISC viruses can provide the sort of immune responsetraditionally obtainable from live virus vaccines, but without thedeleterious side effects that live attenuated viruses pose, such asresidual pathogenicity and reversion to virulence.

The virus may be derived from herpes simplex virus (HSV) in which, forexample, the gene encoding glycoprotein H (gH) has been inactivated ordeleted. The mutant virus may also comprise a heterologous sequenceencoding an immunogen derived from a pathogen. The host cell willsuitably be a recombinant eukaryotic cell line containing the geneencoding HSV glycoprotein H. As another example the virus may be derivedfrom an orthopox virus, for example, vaccinia virus, which again maycomprise a heterologous sequence encoding an immunogen derived from apathogen.

The general teaching hereof may be exemplified by (i) the creation of acell line expressing the HSV-1 gH glycoprotein gene (a gH+ complementingcell line): (ii) the production of HSV-1 virus with an interrupted gHgene (an HSV-1 gH-virus) and carrying a heterologous gene(beta-galactosidase); (iii) the growth of the HSV-1 gH− virus in the gH+complementing cell line. It is shown herein that in experimentsinvestigating the ability of HSV-1 gH-virus and killed HSV-1 to protectagainst infection with wild-type HSV-1, the HSV-1 gH-virus provided goodprotection.

The present application provides in certain examples mutant type-2 HSV(HSV-2 virus) for use as a vaccine, whose genome is defective in respectof a gene essential for the production of infectious HSV-2 such that thevirus can infect normal cells and replicate therein to give rise to theproduction and release from the cells of non-infectious viral particles.The defect can be such that the gH gene encoding the gH protein which isessential for the production of infectious virus has been deleted orinactivated; this mutant HSV-2 defect allows the production and releasefrom the cells of non-infectious virus particles. Such mutant HSV-2virus can be grown in a cell which has a heterologous nucleotidesequence which allows said cell to express the essential gH proteinencoded by said deleted or inactivated gH gene. Such mutant type-2 HSVcan infect normal cells and undergo replication and expression of viralantigens in those cells but cannot produce normal infectious virus. Suchmutant virus can be used prophylactically or therapeutically ingenerating an immune response in a subject infected with HSV eg withHSV-2.

This invention shows a unique way of combining the efficacy and safetyof a killed vaccine with the extra immunological response induced by thein-vivo production of viral protein by the attenuated vaccine. Inpreferred embodiments it comprises two features. Firstly, a selectedgene is inactivated within the virus genome, usually by creating aspecific deletion. This gene will be involved in the production ofinfectious virus, but preferably not preventing replication of the viralgenome. Thus the infected cell can produce more viral protein from thereplicated genetic material, and in some cases new virus particles maybe produced, but these would not be infectious. This means that theviral infection cannot spread from the site of the inoculation.

A second feature of the invention is a cell which provides the viruswith the product of the deleted gene, thus making it possible to growthe virus in tissue culture. Hence, although the virus lacks a geneencoding an essential protein, if it is grown in the appropriate hostcell, it will multiply and produce complete virus particles which are tooutward appearances indistinguishable from the original virus. Thismutant virus preparation is inactive in the sense that it has adefective genome and cannot produce infectious virus in a normal host,and so may be administered safely in the quantity required to generatedirectly a humoral response in the host. Thus, the mutant virus need notbe infectious for the cells of the host to be protected and merelyoperates in much the same way as a conventional killed or attenuatedvirus vaccine. However, preferably the immunising virus is itself stillinfectious, in the sense that it can bind to a cell, enter it, andinitiate the viral replication cycle and is therefore capable ofinitiating an infection within a host cell of the species to beprotected, and producing therein some virus antigen. There is thus theadditional opportunity to stimulate the cellular arm of the host immunesystem.

The deleted or inactivated gene is preferably one involved as late aspossible in the viral cycle, so as to provide as many viral proteins aspossible in vivo for generating an immunogenic response. For example,the gene may be one involved in packaging or some other post-replicativeevent, such as the gH glycoprotein of HSV. However, the selected genemay be one involved in the viral genome replication, and the range ofproteins expressed in vivo will depend upon the stage at which that geneis normally expressed. In the case of human cytomegalovirus (HCMV) theselected gene may be one (other than the Immediate Early gene) thateffectively prevents viral genome replication in vivo, since theImmediate Early gene which is produced prior to viral genome replication(and indeed is essential for it) is highly immunogenic.

This invention can be applied to any virus where one or more essentialgene(s) can be identified and deleted from or inactivated within thevirus genome. For DNA viruses, such as Adeno, Herpes, Papova, Papillomaand Parvo viruses, this can be achieved directly by (i) the in vitromanipulation of cloned DNA copies of the selected essential gene tocreate specific DNA changes; and (ii) re-introduction of the alteredversion into the virus genome through standard procedures orrecombination and marker rescue. The invention however, is alsoapplicable to RNA viruses. Techniques are now available which allowcomplementary DNA copies of a RNA virus genome to be manipulated invitro by standard genetic techniques, and then converted to RNA by invitro transcription. The resulting RNAs may then be re-introduced intothe virus genome. The technique has been used to create specific changesin the genome of both positive and negative stranded RNA viruses, e.g.poliovirus (V R Racaniello and D Baltimore, Science 214 (1981) 916-919)and influenza virus (W Luytjes et al, Cell 59 (1989) 1107-1113).

In theory, any gene encoding an essential protein should be a potentialtarget for this approach to the creation of attenuated viruses. Inpractice however, the selection of the gene will be driven by a numberof considerations.

-   1. The gene should preferably be one which is required later in    infection.

Thus replication of the attenuated virus is not interrupted in the earlyphase. This means that most and possibly all other virus antigens willbe produced in the infected cell, and presented to the host immunesystem in conjunction with host cell MHC class 1 molecules. Suchpresentation leads to the development of cellular immunity against virusinfection through the production of cytotoxic T cells. The cytotoxic Tcells can recognise these antigens, and therefore kill virus infectedcells. It is possible that the deleted gene could represent one which isnot required at all for virus assembly, but is necessary for theassembled virus to be able to infect new cells. An example of such aprotein is the HSV gH protein. In the absence of this protein, HSVvirions are still produced, but they are non-infectious.

-   2. Ideally, the product of the selected gene should not, on its own,    be toxic to the eukaryotic cell, so that a complementing cell can be    produced relatively easily. This however is not an absolute    requirement, since the gene may be placed under the control of an    inducible promoter in the complementing cell, such that its    expression may be switched on only when required.

The nature of the mutation created in the target gene is also a matterof choice. Any change which produces a non-functional gene product issatisfactory, as long as the risk of reversion of a wild type structureis minimised. Such changes include interruption of the target withextraneous sequences and creation of specific deletions. The mostsatisfactory strategy for a vaccine to be used as a therapeutic and/orprophylactic however, would be one where a deletion is made thatencompasses the entire sequence to be introduced into the complementingcell. The approach minimises the risk of regenerating wild type virusthrough recombination between the virus and cell DNA in thecomplementing cell.

Although there are several examples of combinations of specificallyinactivated viruses and complementing cells, (see earlier discussion),to date, these have been used either for basic research on the virus,or, as in the case of retroviruses, to make a safer vector for producingtransgenic animals. They have not been used for vaccine purposes, and tothe applicants knowledge no suggestion of this kind of use has beenproposed.

As well as using such an inactivated virus/complementing cellcombination to produce safe vaccines against the wild-type virus, thisinvention also deals with the use of the same system to produce safeviral vectors for use as vaccines against foreign pathogens.

An example of such a vector is one based on HSV. The HSV genome is largeenough to accommodate considerable additional genetic information andseveral examples of recombinant HSV viruses carrying and expressingforeign genetic material have been described (e.g. M W Ligas and D CJohnson, J Virol 62 (1988) 1486-1494, op. cit.). Thus a virus with adeletion in an essential virus gene as described above, and alsocarrying out and expressing a defined foreign gene, could be used as asafe vector for vaccination to generate an immune response against theforeign protein.

A particular characteristic of HSV is that it may become latent inneurones of infected individuals, and occasionally reactivate leading toa local lesion. Thus an HSV with a deletion in an essential virus geneand expressing a foreign gene could be used to produce deliberatelylatent infection of neurones in the treated individual. Reactivation ofsuch a latent infection would not lead to the production of a lesion,since the virus vector would be unable to replicate fully, but wouldresult in the onset of the initial part of the virus replication cycle.During this time expression of the foreign antigen could occur, leadingto the generation of immune response. In a situation where the deletedHSV gene specified a protein which was not needed for virus assembly,but only for infectivity or assembled virions, such a foreign antigenmight be incorporated into the assembled virus particles, leading toenhancement of its immunogenic effect. This expression of the foreigngene and incorporation of its protein in a viral particle could ofcourse also occur at the stage where the mutant virus is first producedin its complementing host, in which case the mutant virus when used as avaccine could present immediately the foreign protein to the speciesbeing treated.

In another example, vaccinia virus, a poxvirus, can carry and expressgenes from various pathogens, and it has been demonstrated that theseform effective vaccines when used in animal experimental systems. Thepotential for use in humans is vast, but because of the known sideeffects associated with the widespread use of vaccinia as a vaccineagainst smallpox, there is reluctance to use an unmodified vacciniavirus on a large scale in humans. There have been attempts to attenuatevaccinia virus by deleting non-essential genes such as the vacciniagrowth factor gene (R M L Buller, S Chakrabarti, J A Cooper, D RTwardzik and B Moss, J Virology 62 (1988), 866-874). However, suchattenuated viruses can still replicate in vivo, albeit at a reducedlevel. No vaccinia virus with a deletion in an essential gene has yetbeen produced, but such a virus, deleted in an essential gene asdescribed above, with its complementing cell for growth, would provide asafer version of this vaccine vector.

A further advantage of this general strategy for immunisation againstheterologous proteins is that it may be possible to perform multipleeffective vaccinations with the same virus vector in a way not possiblewith conventional live virus vectors. Since a standard live virusvaccine probably relies for its efficacy on its ability to replicate inthe host animal through many cycles of infection, its usefulness will beseverely curtailed in an individual with immunity against that virus.Thus a second challenge with the same virus, whether to provide abooster immunisation against the same protein, or a new response againsta different protein, is likely to be ineffective. Using a virus vectorwith a deletion in an essential gene however, where multi-cyclereplication is not desired or required, the events leading to effectiveimmunisation will occur very soon after immunisation. The dose of themutant virus can be relatively large (since it should be completelysafe), and it is therefore unlikely that these early events will beblocked by the host immune response, which will require some time to bemobilised completely.

Although we have referred above to a mutant virus being defective in anessential gene, and optionally containing a gene for an immunogenicpathogen protein, the mutant could be defective in more than oneessential gene, and/or contain more than one immunogenic pathogenprotein gene. Thus, the mutant virus might include the gene for HIV gp120, to act as a vaccine in the manner suggested above, and also thegene for the HSV gag protein to be expressed within the vaccinated hostand presented at the surface of the host cell in conjunction with MHC-Ito stimulate a T-cell response in the host.

The present invention also provides a pharmaceutical preparation whichcomprises a mutant non-retroviral virus whose genome is defective inrespect of a gene essential for the production of infectious virus suchthat the virus can infect normal cells and undergo replication andexpression of viral antigen genes in those cells but cannot producenormal infectious virus, for prophylactic or therapeutic use ingenerating an immune response in a subject infected therewith.

The mutant virus of the pharmaceutical preparation can be a mutantnon-retroviral virus whose genome is defective in respect of a geneessential for the production of infectious virus such that the virus caninfect normal cells and replicate therein to give rise to the productionand release from the cells of non-infectious viral particles. Thepharmaceutical can be a vaccine capable of protecting a patientimmunised therewith against infection or the consequences of infectionby a non-retroviral virus. The pharmaceutical can be a vaccine capableof protecting a patient immunised therewith against infection or theconsequences of infection by the corresponding wild-type virus.

The pharmaceutical can be a therapeutic capable of treating a patientwith an established non-retroviral virus infection, e.g. an infectionestablished by the corresponding wild-type virus.

The pharmaceutical can be adminstrable sub-cutaneously,intra-muscularly, intra-dermally, epithelially-, (with or withoutscarification), nasally-, vaginally-, or orally- and can compriseexcipient(s) suitable for the selected administration route.

The mutant virus contained in the pharmaceutical preparation can becapable of protecting a patient immunised therewith against infection orthe consequences of infection with HSV eg infection by the correspondingwild-type virus.

The present invention also provides use of a mutant type-1 HSV whosegenome is defective in respect of a gene essential for the production ofHSV-1 such that the virus can infect normal cells and undergoreplication and expression of viral antigen genes in those cells butcannot produce normal infectious virus, for preparation of apharmaceutical for prophylactic or therapeutic use in generating animmune response in a subject against type-2 HSV infection.

The use may be in respect of pharmaceuticals for intra-epithelial (withor without scarification), intra-vaginal, intra-nasal or per-oraladministration.

The present invention also provides an assembly comprising apharmaceutical (for prophylaxis ie a vaccine or for therapy ie atherapeutic) as described above in a container preferably a pre-filledsyringe or glass vial/ampoule with printed instructions on oraccompanying the container concerning the administration of thepharmaceutical to a patient to prevent or treat conditions caused byinfection with a non-retroviral virus, e.g. HSV infection by HSV-1and/or HSV-2. The printed instructions may concern the prevention ortreatment of facial or genital lesions.

Vaccines containing the mutants as described can be prepared inaccordance with methods well known in the art wherein the mutant iscombined in admixture with a suitable vehicle. Suitable vehiclesinclude, for example, saline solutions, or other additives recognised inthe art for use in compositions applied to prevent viral infections.Such vaccines will contain an effective amount of the mutant as herebyprovided and a suitable amount of vehicle in order to prepare a vaccineuseful for effective administration to the host.

Dosage rates can be determined according to known methods.

For example, dosage rate may be determined by measuring the optimumamount of antibodies directed against a mutant resulting fromadministration of varying amounts of the mutant in vaccine preparations.Attention is directed to ‘New Trends and Developments in Vaccines’,editors A Voller and H Friedman, University Park Press, Baltimore, 1978,for further background details on vaccine preparation.

Therapeutics comprising a mutant as herein provided can be formulatedaccording to known methods to provide therapeutically usefulcompositions, whereby the mutant is combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation are described in ‘Remington’s Pharmaceutical Sciences' (MackPublishing Co, Easton, Pa., ed. A R Gennaro), by E W Martin, and by FRola. Such compositions contain an effective amount of the mutant virushereof together with a suitable amount of carrier vehicle in order toprepare therapeutically acceptable compositions suitable for effectiveadministration to the host.

Typically vaccines are prepared as injectables, (traumatic ornon-traumatic) either as liquid solutions or suspensions: solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. Preparations may also be encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, trehalose, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as other stabilisers and/or pH buffering agents, whichenhance the stability and thus the effectiveness of the vaccine.

The vaccines may be administered parenterally, by injection, forexample, subcutaneously, intraepithelially (with or withoutscarification). Additional formulations which are suitable for othermodes of administration eg oral, vaginal and nasal formulations are alsoprovided. Oral formulations include such normally employed excipientsas, for example, pharmaceutical grades of trehalose mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, and the like. The compositions may take the form ofsolutions, suspensions, tablets, pills, capsules sustained releaseformulations or powders.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically effective.The quantity to be administered will have been predetermined frompreclinical and clinical (phase I) studies to provide the optimumimmunological response.

The vaccine may be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-3 separate doses, followedby other doses given at subsequent time intervals required to maintainand or re-enforce the immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, have been determined from preclinical andclinical studies as maintaining the optimum immunological response overtime.

The invention is further described herein by way of example only, andnot by way of limitation, with reference to the following sections ofdetailed description, and to the accompanying Figures.

Sections of detailed description in the present application are:

-   -   A. Generation of a Cell line expressing the HSV type 1 gH gene.    -   B. Production of HSV type 1 virus with an interrupted gH gene.    -   C. Studies on the protective effect of gH-negative HSV compared        to heat killed virus. The data given herein include in-vivo data        which show that intra-epithelial vaccination of mice via the ear        with a gH-negative mutant form of HSV-1 gave better protection        against later challenge with wild-type HSV-1, than similar        vaccination with killed HSV-1. A clear protective effect against        the establishment of latent infection in the cervical ganglia        was also shown for vaccination with the mutant HSV-1.    -   D. HSV lacking the gH gene as a vector for immunisation against        a foreign antigen: introduction of a gp120 gene.    -   E. Preparation of a DISC HSV-2 mutant virus and complementing        gH+ cell line.    -   F,G. Sections F and G of the detailed description show results        indicated as follows:    -   (1) In a study using the mouse ear model the results reported in        section C of the detailed description herein were confirmed.        Intra-epithelial vaccination of mice with DISC HSV-1 led to        complete protection against replication of the challenge virus        wild type (w.t.) HSV-1. Little effective protection was provided        by equivalent doses of inactivated HSV-1. DISC HSV-1 also        protected against the establishment of latent infection in the        cervical ganglia.    -   (2) Also in the mouse ear model it is shown that no significant        differences in antibody titres were observed between animals        vaccinated with DISC HSV-1 and an equivalent amount of        inactivated HSV-1.    -   (3) Also in the mouse ear model it is shown that at low        vaccination doses, inactivated HSV-1 failed to established a        delayed-type hypersensitivity (DTH) response, whilst equivalent        doses of DISC HSV-1 established a DTH response. At high doses,        both DISC HSV-1 and inactivated HSV-1 induced similar DTH        responses.    -   (4) Also in a mouse study it was shown that in contrast to        vaccination with inactivated HSV-1, vaccination with DISC HSV-1        induced HSV-1 specific cytotoxic T cell activity.    -   (5) The in vivo mouse ear model was used to study long term        prophylactic effect of DISC HSV-1. Two vaccinations of DISC        HSV-1 was found to provide better long term protection against        challenge with w.t. HSV-1 than two vaccinations of inactivated        DISC HSV-1    -   (6) The in vivo mouse ear model was used to investigate the        prophylactic effect of DISC HSV-2 against HSV-2 infection.        Intra-epithelial vaccination of mice with DISC HSV-2 provided        better protection against replication of the challenge virus        w.t. HSV-2 than inactivated DISC HSV-2.    -   (7) The in vivo guinea-pig vaginal model was used to study the        prophylactic effect of DISC HSV-1 against HSV-2 infection. It        was shown that intra-epithelial or intra-vaginal vaccination        with DISC HSV-1 provided a high degree of protection against the        primary symptoms of HSV-2 infection. Immunisation with DISC        HSV-1 or inactivated virus retarded growth of challenge virus        w.t. HSV-2 in the vagina. Further intra-vaginal vaccination with        DISC HSV-1 lessened the number of recurrent HSV-2 lesions in a        100 day follow-up period. Intra-epithelial vaccination with DISC        HSV-1 and inactivated virus also resulted in reduced recurrent        lesions, but compared to intra-vaginal vaccination with DISC        HSV-1, the reduction was less.    -   (8) Oral and intranasal vaccination of guinea-pigs with DISC        HSV-1 led to protection against acute disease symptoms following        challenge with w.t. HSV-2. The intranasal route appeared to be        more effective than the oral route.    -   (9) In guinea-pigs which had recovered fully from primary HSV-2        disease, the therapeutic administration of DISC HSV-1 either        intra-vaginally or intra-epithelially resulted in an apparent        reduction in the frequency of recurrent of disease symptoms        compared with mock vaccinated animals. The per vaginum        vaccination route in comparison to oral or intra-nasal        vaccination resulted in significantly lower levels of recovered        virus following challenge.    -   (10) In guinea-pigs which had recovered fully from primary HSV-2        disease, intra-vaginal therapeutic administration of DISC HSV-2        was more effective in reducing the frequency of recurrence of        disease symptoms than treatment with DISC HSV-1.

Referring to the Figures herein:

FIG. 1 illustrates the production of plasmid pGH1.

FIG. 2 illustrates the production of plasmid pGH2.

FIG. 3 a shows the pair of complementary oligonucleotides (SEQ ID NO:1,SEQ ID NO:2) used to generate the plasmid pSP64Ta.

FIG. 3 b illustrates the production of plasmid pSP64TA.

FIG. 4 a shows the two oligonucleotides (SEQ ID NO:3, SEQ ID NO:4) usedto generate the plasmid pCMVIEP.

FIG. 4 b illustrates the plasmid pCMVIEP.

FIG. 5 illustrates the plasmid pCMVlacZ.

FIG. 6 illustrates the plasmid pGH3.

FIG. 7 illustrates the strategy for construction of plasmid pGH-120.

FIG. 8 shows clearance of wild-type HSV-1 (w.t. HSV-1) strain SC16 virusin the ears of mice vaccinated with either live DISC HSV-1 orinactivated (β-propiolactone treated) w.t. HSV-1 (strain SC16). Groupsof 4 mice were vaccinated at the doses indicated by scarification of theleft ear pinna. Mice were challenged 14 days post-vaccination with 2×10⁶pfu w.t. HSV-1 strain SC16 in the right ear pinna and virus titres weremeasured 5 days post challenge. Data are expressed as the geometricmeans and standard errors of the means.

FIG. 9 shows measurement of titres of neutralising and ELISA antibody tow.t. HSV-1 in mice vaccinated with either w.t. HSV-1 (strain SC16), liveDISC HSV-1, killed DISC HSV-1 or PBS. Sera from mice were assayed in thepresence of complement for neutralising antibodies to w.t. HSV-1 in aplaque reduction assay. Individual titres are expressed as thereciprocal dilution of sera required to neutralise 50% of theinfectivity obtained in the absence of antibody.

FIG. 10 shows delayed-type hypersensitivity (DTH) responses in micevaccinated with either w.t. HSV-1 (strain SC16), live DISC HSV-1, killedDISC HSV-1 or PBS. Mice were vaccinated in the left ear pinna at thedoses indicated 14 days prior to challenge with 10⁶ pfu w.t. HSV-1(strain SC16) in the opposite ear. Ear thickness was measured 24 and 48hours post-challenge and is expressed as the difference between thechallenged and vaccinated ear. Data are presented as the means ofdifferences in ear thickness (in μm).

FIG. 11 shows cytotoxic T cell (CTL) responses in mice vaccinated witheither live DISC HSV-1, killed DISC HSV-1, MDK (a thymidine kinasenegative HSV-1 strain) or PBS. Mice were immunised twiceintraperitoneally three weeks apart and cell suspensions made fromspleens 10 days after the second injection. Cells were stimulated invitro for 4 days before being tested in a CTL assay using ⁵¹Cr-labelledA20/2J as target cells. Data are presented as mean % ⁵¹Cr release fromquadruplicate samples at each point. Standard errors of the means areall <10%.

FIG. 12 shows clinical symptoms as assessed by erythema score inguinea-pigs post challenge with 10^(5.2) pfu w.t. HSV-2 (strain MS)subsequent to vaccination with doses of 2×10⁷ pfu DISC HSV-1 at a 3 weekinterval either by the intra-epithelial or the intra-vaginal route;

FIG. 13 shows clinical symptoms as assessed by total lesion score inguinea-pigs post challenge with 10^(5.2) pfu w.t. HSV-2 (strain MS)subsequent to vaccination with doses of 2×10⁷ pfu DISC HSV-1 at a 3 weekinterval either by the intra-epithelial or the intra-vaginal route.

FIG. 14 shows post challenge virus w.t. HSV-2 (strain MS) replication inguinea-pigs post challenge with 10^(5.2) pfu w.t. HSV-2 (strain MS)subsequent to vaccination with doses of 2×10⁷ pfu DISC HSV-1 at a 3 weekinterval either by the intra-epithelial or the intra-vaginal route.

FIGS. 15 a and 15 b show recurrent disease in guinea-pigs post challengewith 10^(5.2) pfu w.t. HSV-2 (strain MS) subsequent to vaccination withdoses of 2×10⁷ pfu DISC HSV-1 at a 3 week interval by theintra-epithelial or the intra-vaginal route. FIG. 15 a shows recurrentdisease as the cumulative mean erythema index per animal. FIG. 15 bshows recurrent disease as cumulative mean number of days with diseaseper animal.

FIG. 16 shows mean lesion score per animal (guinea-pigs) with w.t. HSV-2(strain MS) infection and which have been vaccinated via the vaginal,oral or nasal routes with a mock virus preparation, DISC HSV-1 orinactivated DISC HSV-1.

FIG. 17 shows mean erythema score per animal (guinea-pigs) with w.t.HSV-2 (strain MS) infection and which have been vaccinated via thevaginal, oral or nasal routes with a mock virus preparation, DISC HSV-1or inactivated DISC HSV-1.

FIG. 18 shows the mean log titre of w.t. HSV-2 (strain MS) per animal(guinea-pigs) with w.t. HSV-2 (strain MS) infection and which have beenvaccinated via the vaginal, oral or nasal routes with a mock viruspreparation, DISC HSV-1 or inactivated DISC HSV-1.

FIG. 19 shows recurrent disease following therapeutic vaccination. Thisis shown as mean cumulative number of days on which disease was observed(disease/days) in groups of guinea-pigs vaccinated with DISC HSV-1either intra-epithelially or intra-vaginally or with a mock viruspreparation intra-vaginally after challenge with w.t. HSV-2 (strain MS).Disease was classified as either presence of one or more lesions or anerythema score of 1 or more. Animals were monitored from 4 weeks afterinitial challenge with w.t. HSV-2 (strain MS) (day o) for 100 days.Animals were vaccinated at Day 0, Day 24 and Day 44 with 2×10⁷ pfu orequivalent dose as indicated.

FIG. 20 relates to the long-term protective effect in mice ofvaccination with DISC HSV-1 against challenge with w.t. HSV-1 (strainSC16). The graph shows the mean log titre of w.t. HSV-1 in the ears 5days post challenge and 223 days post vaccination.

FIG. 21 relates to the long-term protective effect in mice ofvaccination with DISC HSV-1 against challenge with w.t. HSV-1 (strainSC16). The graph shows neutralising antibody titres days 15, 27, 90, 152and 218 post vaccination as stated.

FIG. 22 relates to the protective effect in mice of vaccination withDISC HSV-2 against challenge with w.t. HSV-2 (strain HG52) forvaccinations with live DISC HSV-2, killed DISC HSV-2 and w.t. HSV-2(strain HG52) at varying doses, the graph shows mean log titre of w.t.HSV-2 in the ear post challenge.

FIG. 23 illustrates the construction of a single plasmid containing thecomplete HSV-2 gH gene.

FIG. 24 shows the sequence (SEQ ID NO:5) of HSV-2 strain 25766 in theregion of the gH gene including a translation of the gH gene in singleletter amino acid code (SEQ ID NO:6).

FIG. 25 shows a comparison of the DNA sequence of HSV-1 (SEQ ID NO:7)and HSV-2 strain 25766 (SEQ ID NO:6) in the region of the gH gene.

FIG. 26 shows a comparison of the deduced amino acid sequences of theHSV-1 strain 17 (SEQ ID NO:8) an HSV-2 strain 25766 (SEQ ID NO:6) gHproteins.

FIG. 27 shows graphically the level of similarity between the DNAsequences of HSV-1 and HSV-2 (SEQ ID NO:5) in the region of the gH gene(from UWGCG program Plotsimilarity).

FIG. 28 shows graphically the level of similarity between the amino acidsequences of the HSV-1 (SEQ ID NO:8) and HSV-2 (SEQ ID NO:6) gH proteins(from UWGCG program Plotsimilarity).

FIG. 29 shows the construction of pIMMB26; two fragments from the leftand right sides of the HSV2 gH gene were amplified by PCR and clonedinto pUC119. The four oligonucleotides MB57 (SEQ ID NO:9), MB58 (SEQ IDNO:10), B59 (SEQ ID NO:11), MB60 are shown.

FIG. 30 shows the construction of pIMMB45.

FIG. 31 shows construction of the first stage recombination vectorpIMMB47+.

FIG. 32 shows construction of the second stage recombination vectorpIMMB46.

FIG. 33 shows a restriction map analysis for recombinants HG52-D, TKminus DISC virus, TK plus DISC virus.

FIG. 34 shows Southern blots of BamHI digestions of various viruses,probed with the right-hand flanking sequence as shown in FIG. 33. Lane5: HG52-D virus, lane 2: TK-minus “first stage” DISC virus and lanes 3,4, 6, 7 and 8: TK-plus “second stage” DISC viruses.

FIG. 35 illustrates diagrammatically the DISC virus concept.

HERPES SIMPLEX VIRUS DELETED IN GLYCOPROTEIN H GENE (gH− HSV)

Herpes simplex virus (HSV) is a large DNA virus which causes a widerange of pathogenic symptoms in man, including recurrent facial andgenital lesions, and a rare though often fatal encephalitis. Infectionwith this virus can be controlled to some extent by chemotherapy usingthe drug Acyclovir, but as yet there is no vaccine available to preventprimary infection. A difficulty with vaccination against HSV is that thevirus generally spreads within the body by direct transfer from cell tocell. Thus humoral immunity is unlikely to be effective, sincecirculating antibody can only neutralise extracelluar virus. Of moreimportance for the control of virus infection, is cellular immunity, andso a vaccine which is capable of generating both humoral and cellularimmunity, but which is also safe, would be a considerable advantage.

A suitable target gene for inactivation within the HSV genome is theglycoprotein H gene (gH). The gH protein is a glycoprotein which ispresent on the surface of the virus envelope. This protein is thought tobe involved in the process of membrane fusion during entry of the virusinto the infected cell. This is because temperature sensitive virusmutants with a lesion in this gene are not excreted from virus infectedcells at the non-permissive temperature (P J Desai et al, J Gen Virol 69(1988), 1147-1156). The protein is expressed late in infection, and soin its absence, a considerable amount of virus protein synthesis maystill occur.

All procedures are carried out using standard procedures in the art, inparticular genetic manipulation procedures are carried out according tomethods described in “Molecular Cloning, A Laboratory Manual”, eds.Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press,1989.

The present description refers to certain strains of HSV-1 and HSV-2. Itis not necessary that the description contained herein is put intoeffect with precisely the mentioned strains. Strains of HSV-1 and HSV-2having high sequence homology to one another by which the invention maybe put into effect are readily available. For example, one source of HSVis the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 USA. The following are available from ATCC underthe indicated accession numbers.

HSV-1 strain F ATCC accession no. VR-733 HSV-1 strain MacIntyre ATCCaccession no. VR-539 HSV-1 strain MP ATCC accession no. VR-735 HSV-2strain G ATCC accession no. VR-734 HSV-2 strain MS ATCC accession no.VR-540Section A. Generation of a Cell Line Expressing the HSV Type 1 gH Gene

The gH gene is present in the Unique Long region (U_(L)) of the HSV type1 genome, between nucleotides 46382 and 43868 (DJ McGeoch et al, J GenVirol 69 (1988), 1531-1574). A cloned copy of this gene is availablewithin the plasmid pAF2. This plasmid was produced by excising aBgIII-Xhol fragment, encompassing the complete gH coding sequence, fromthe plasmid pTZgH, and cloning it into the BgIII site of plasmid pSP64Tas described (U A Gompels and A C Minson. J Virol, 63 (1989),4744-4755). A HindIII fragment containing the promoter sequence for theglycoprotein D (gD) gene (extending from nucleotides −392 to +11, withrespect to the start of the gD gene) is then excised from the plasmidpSVD4 (R D Everett, Nucl Acids Res, 11 (1983), 6647-6666), and clonedinto the unique HindIII site of pAF2 to generate pGH1 (FIG. 1) such thatthe promoter sequence is in the correct orientation to drive expressionof the gH gene. Thus this plasmid contains the complete gH codingsequence under the control of the HSV type 1 gD gene promoter. Thisplasmid is then purified and then co-transfected into Vero cells withthe plasmid pNE0 (from Pharmacia LKB Biotechnology Inc.) using thestandard calcium phosphate co-precipitation technique (F L Graham and AJ Van der Eb, Virology 52 (1973), 456-467).

Vero cells which have acquired resistance to neomycin are then selectedby passage of the cells in the drug G418, and colonies of these cellscloned by limiting dilution. These neomycin resistant cells are thenamplified in tissue culture, and samples are then infected with HSV type2 virus. Infection with the HSV type 2 virus has the effect of inducingtranscription from the type 1 gD promoter present in the complementingcell genome, and so of stimulating production of the type 1 gH proteinin the complementing cell. Lysates of the infected cells are thenscreened for expression of the gH protein by western blotting, using apolyclonal antiserum known to recognise specifically the type 1 gHprotein (P J Desai et al, J gen Virol 69 (1988) 1147-1156, op. cit.).Cells which express the required protein are then retained and frozenstocks prepared. This material represents the gH+ complementing cellline.

Section B. Production of HSV Type 1 Virus with an Interrupted gH Gene

A 6432 base pair BgIII fragment containing the coding sequence of gHtogether with HSV flanking sequences is excised from the plasmid pUG102(U A Gompels and A C Minson, Virology 153 (1986), 230-247) and clonedinto the plasmid pAT153 (A J Twigg and D Sherrat, Nature, 283 (1980),216-218) to generate pGH2 (FIG. 2). This plasmid is digested with PvuIIwhich cuts only within the gH coding sequence at two positions(nucleotides 44955 and 46065 according to the numbering scheme of D JMcGeoch et al, 1988, op cit.), and the larger of the two fragmentspurified. A fragment of DNA consisting of the complete B-galactosidasegene from E coli downstream of the Immediate Early gene promoter fromCytomegalovirus (CMV) is then prepared by the following procedure. Firstof all a pair of complementary oligonucleotides (SEQ ID NO:1, SEQ IDNO:2) (shown in FIG. 3 a) are annealed and ligated with BgIII-digested,phosphatase-treated pSP64T (P A Krieg and D A Melton, Nucl Acids Res 12(1984), 7057-7070) to generate the plasmid pSP64Ta as shown in FIG. 3 b.The added linker also includes the initiation codon and first threecodons of the B-galactosidase gene (lacZ) of E. coli. Next the “coreregion” of the Immediate Early gene promoter of CMV is amplified fromplasmid pUG-H1 (U A Gompels and A C Minson, 1989, op. cit.) by thePolymerase Chain Reaction technique (PCR—Molecular Cloning, ed. Sambrooket al., op cit.) using the two oligonucleotides (SEQ ID NO:3, SEQ IDNO:4) shown in FIG. 4 a, which correspond to sequences from −302 to −288(SEQ ID NO:3) from −13 to −36 (SEQ ID NO:4) respectively (numbered inrelation to the start of the CMV Immediate Early gene as described by AAkrigg et al, Virus Research, 2 (1985), 107-121). These oligonucleotides(SEQ ID NO:3, SEQ ID NO:4) also contain, at their 5′ ends, sites for therestriction enzyme HindIII, and in the case of the oligonucleotideannealing upstream of the promoter (SEQ ID NO:3), an additional SmaIsite. The PCR-amplified product DNA is then digested with HindIII, andcloned into HindIII-digested pSP64Ta, to generate the plasmid pCMVIEP(FIG. 4 b). Finally, a DNA fragment containing a complete copy of the E.coli B-galactosidase gene, lacking only the extreme 5′ end of the codingsequence, is isolated by digestion of the plasmid pSC8 (S Chakrabarti etal, Mol Cell Biol, 5 (1985), 3403-3409) with BamHI, and cloned into theunique BgIII site of pCMVIEP to generate pCMVlacZ (FIG. 5). A fragmentof DNA containing the B-galactosidase gene under the control of the CMVIE promoter is then isolated by digestion of pCMVlacZ with SmaI, andligated with the purified PvuII fragment of pGH2 described above, togenerate pGH3, which consists of a copy of the gH gene interrupted by afunctional B-galactosidase gene (FIG. 6).

The next step is to replace the wild type gH gene in the HSV genome withthis interrupted version, and this is done by allowing recombinationbetween HSV DNA and plasmid pGH3, followed by selection of those viruseswhich have acquired a functional B-galactosidase gene. Plasmid pGH3 DNAis therefore cotransfected into cells expressing the gH gene (the gH+complementing cell line described in section A) along with purified HSVDNA isolated from purified HSV virions (R A Killington and K L Powell,in “Growth, Assay and Purification of Heprpesviruses”, ch. 10 in“Techniques in Virology: A practical Approach” (ed. B W J Mahy) pp207-236, IRL Press, Oxford, 1985) by the standard calcium phosphateprecipitation technique (F L Graham and A J Van der Eb, 1973, op. cit.).

The progeny HSV virus produced from this transfection experiment is thenplated on monolayers of gH+ complementing cells by standard plaqueassay, using an agar overlay, in the presence of5-bromo-chloro-3-indolyl-β-D-galactoside (X-gal), a chromogenicsubstrate which is converted to a blue substance by the enzymeβ-galactosidase. Thus plaques resulting from infection by virus genomescontaining and expressing the β-galactosidase gene will appear blue.These virus genomes should therefore carry an interrupted version of thegH gene. Virus is recovered from these plaques by picking plugs of agarfrom the appropriate part of the plate, and virus stocks preparedthrough growth of virus in the gH+ complementing cell line. Theseviruses, since they bear non-functional versions of the gH gene, shouldbe unable to form plaques on cells which do not contain and express anendogenous functional copy of the gH gene, and so to confirm this, asample of the virus is assayed for its ability to form plaques on wildtype Vero cell monolayers in comparison with the gH-complementing cells.

Finally, virus DNA is prepared from these stocks, and checked for theexpected DNA structure around the gH gene by Southern blotting. Afterconfirmation of the correct genetic structure, a large stock of the gHgene-deficient virus is then prepared by inoculation of a sample of thevirus into a large-scale culture of the gH+ complementing cell line(multiplicity of infection=0.01), and three days later, the infectedcells are harvested. The infected cells are disrupted by sonication inorder to release the cell-associated virus, and the total sonicatedmixture stored at −70° as the virus master stock. The titre of the virusas the stock is then established by plaque assay on the gH+complementing cell line. Samples of this virus stock are then used toprepare working stocks as before, and these working stocks are then usedto infect laboratory animals as described below.

Publications relevant to these mutant viruses are A Forrester et al, JVirol, 66 (1992), pp 341-348, and H E Farrell et al, J Virol 68 (1994),pp 927-932.

Section C. Studies on the Protective Effect of gH-Negative HSV Comparedto Heat Killed Virus

In order to assess the host immunological response to this virus,challenge experiments were conducted in mice according to theexperimental plan described below.

The protective effect of a live gH virus preparation was compared withan inactivated preparation of wild type (WT) virus (strain SC16) asfollows.

Preparation of Inactivated Wild Type Virus for Vaccination:

HSV type 1 (strain SC16) was grown by low multiplicity infection (0.01pfu/cell) of Vero cells. After three days, the virus was harvested, andcytoplasmic virus recovered by Dounce homogenisation. Nuclei wereremoved by centrifugation at 500×g for 15 min, and the virus wasrecovered from the supernatant by centrifugation on to a 40% sucrosecushion at 12K for 60 min Beckman Sw27 rotor. The banded virus wasdiluted, pelleted and purified by sucrose gradient centrifugation(Killington and Powell, 1985, op. cit.). The virus band was harvestedfrom the gradient, and the virus recovered by centrifugation. Virus wasresuspended in phosphate-buffered saline (PBS), assayed for infectivityby plaque titration on baby hamster kidney (BHK) cells, and the particlecount determined by electron microscopy. The particle:infectivity ratioof the preparation was 110 particles/pfu. The virus was diluted to2.5×10¹⁰ pfu/ml in PBS, and inactivated by treatment withβ-propiolactone for 60 min at 20° C. Aliquots were then stored at −70°C.

Preparation of Live gH⁻ Virus for Vaccination:

This material was prepared as described for the wild type virus, exceptthat the virus was grown in the gH+ complementing cell line containingand expressing the HSV type 1 gH gene, and it was not inactivated bytreatment with β-propiolactone. The particle:infectivity ratio of thispreparation was 150:1. The concentration of this preparation wasadjusted to 2.5×10¹⁰ pfu/ml and aliquots were stored in PBS at −70° C.

Vaccination Protocol:

4 week-old female balb/C mice (purchased from Tucks U.K. Ltd) werevaccinated with various doses of inactivated WT virus or live gH− virusin 2 μl volumes of phosphate-buffered saline by droplet application andneedle scarification of the right ear as follows:

Group A Control - no virus Group B 5 × 10⁴ pfu virus vaccine Group C 5 ×10⁵ pfu virus vaccine Group D 5 × 10⁶ pfu virus vaccine Group E 5 × 10⁷pfu virus vaccine

After 14 days, all mice were challenged by similar inoculation of theleft ear with 2×10⁶ pfu HSV-1 strain SC16 (wild type virus). Mice werekilled after 5 days and assayed for virus infectivity in the left earand left cervical ganglia cII, cIII and cIV (combined). For latencystudies, other vaccinated and challenged animals were killed after 1month, and tested for latent infection by dissecting out the cII, cIIIand cIV ganglia. These were incubated in medium for five days thenhomogenised and assayed for the presence of infectious virus by standardplaque assay. All the following results are expressed as pfu/organ.

TABLE C-1 Titre of challenge virus present during the acute phase ofinfection after vaccination with live gH- virus Virus titre - log₁₀pfu(WT SC16) cervical Mouse no. Ears mean ganglia* mean Group A 1 4.2 4.33.3 3.4 2 4.2 3.4 3 4.6 3.4 4 4.3 3.4 Group B 1 3.4 0.85 1.5 1.8 2 none2.4 3 none 2.0 4 none 1.5 Group C 1 none — none — 2 none none 3 nonenone 4 none none Group D 1 none — none — 2 none none 3 none none 4 nonenone Group E 1 none — none — 2 none none 3 none none 4 none none *Pooledcervical ganglia cII, cIII and cIV

TABLE C-2 Titre of challenge virus present during the acute phase ofinfection after vaccination with inactivated WT HSV-1 Virus titre -log₁₀pfu (WT SC16) cervical Mouse no. Ears mean ganglia* mean Group A 15.7 5.2 2.6 2.3 2 4.4 2.3 3 5.7 2.1 Group B 1 4.2 3.8 1.9 1.2 2 3.6 3.13 3.5 none 4 3.8 none Group C 1 none 2.0 none — 2 2.5 none 3 2.9 none 42.7 none Group D 1 3.9 2.6 none — 2 2.0 none 3 2.0 none 4 2.3 none GroupE 1 none — none — 2 none none 3 none none 4 none none *Pooled cervicalganglia cII, cIII and cIV

TABLE C-3 Titre of challenge virus present as latent virus in thecervical ganglia after vaccination with live gH-HSV-1 Virus titre incervical ganglia* Reactivation Mouse No. (log₁₀pfu WT) frequency Group A1 5.4 5/5 2 4.6 3 5.0 4 4.8 5 5.3 Group B 1 none 3/4 2 1.5 3 5.1 4 5.3Group C 1 none 1/3 2 none 3 3.2 Group D 1 none 0/4 2 none 3 none 4 noneGroup E 1 none 0/4 2 none 3 none 4 none *Pooled cervical ganglia cII,cIII and cIV

TABLE C-4 Titre of latent challenge virus in the cervical ganglia aftervaccination with inactivated WT HSV-1 Virus titre in cervical ganglia*Reactivation Mouse No. (log₁₀pfu WT) frequency Group A 1 none 3/4 2 5.03 5.0 4 5.2 Group B 1 3.5 3/4 2 4.0 3 5.5 4 none Group C 1 3.6 2/4 2 5.13 none 4 none Group D 1 none 1/4 2 4.8 3 none 4 none Group E 1 none 0/42 none 3 none 4 none *Pooled cervical ganglia cII, cIII and cIV (p.f.u.= plaque forming units; gH- is a virus with a defective gH gene).

These results show the titre of the challenge virus wt SC16 present inthe ears and cervical ganglia during the acute phase of infection. Thus,a low titre indicates good effectiveness of the vaccination regimen withgH− virus whereas a higher titre, indicates poorer effectiveness. It isclear from the results that vaccination with live gH− HSV virus is verymuch more effective than an equivalent amount of inactivated WT virus.With the inactivated preparation, a dose of 5×10⁷ pfu was required toprevent challenge virus replication in the ear, whereas with the livegH− virus; 100-1000 fold less virus was required. Live gH− virusvaccination with 5×10⁵ pfu and over, was also able to block replicationof the challenge virus in the cervical ganglia during the acute phase ofinfection, and furthermore showed a clear protective effect against theestablishment of latent infection in the cervical ganglia.

Section D. HSV Lacking the gH Gene as a Vector for Immunisation Againsta Foreign Antigen: Introduction of the gp120 Gene of SIVmac Strain 142into the Genome of gH− HSV Virus

Viruses with deletions in essential genes may, as described above, beused as safe vectors for the delivery of foreign antigens to the immunesystem, and the gH− HSV virus described above provides a suitableexample of a such a vector. This virus could be used to express anydesired foreign antigen, but a particularly attractive possibility wouldbe the major antigenic proteins of the AIDS virus human immunodeficiencyvirus (HIV). Thus these sequences would be inserted into the gH− HSVgenome in a way that would ensure their expression during infection ofnormal cells (i.e. non-complementing cells) by the recombinant virus.Infection of an individual with such a virus could lead to a latentinfection which, from time to time upon reactivation, would lead to aburst of production of the foreign antigen, resulting in stimulation ofthe immune response to that protein.

Since studies to test this approach directly in humans are not feasibleat present, as an initial stage, the approach may be tested in monkeysusing the Simian AIDS virus SIV_(mac) (Simian immunodeficiency virusisolated from macaques). A suitable SIV gene for this purpose is thatencoding the gp120 protein, one of the major antigenic targets for thisvirus. This gene is therefore introduced into the gH− HSV genome, andthe efficacy of this virus as a vaccine to protect monkeys againstchallenge with SIV assessed.

The SIV gp120 gene is first of all cloned next to the cytomegalovirus IEcore promoter (U A Gompels and A C Minson, 1989, op. cit.), andsubsequently a DNA cassette consisting of the gp120 gene and theupstream CMV promoter is cloned into plasmid pGH2 (FIG. 2). Theresulting plasmid is then co-transfected into the gH+ complementing cellline along with DNA purified from the gH− HSV, and recombinant viruswhich has acquired the gp120 gene in place of the β-galactosidase genepresent in the gH− HSV virus is isolated by screening for interruptionof the β-galactosidase gene.

D-A. Construction of Plasmid for Recombinant of the SIV gp120 CodingSequence into the HSV Genome

The overall scheme for this procedure is shown in FIG. 7. A SacIrestriction enzyme fragment (corresponding to bases 5240-8721) isexcised from a cloned DNA copy of the SIV genome (L Chakrabarti et al,Nature 328 (1987), 543-547), and cloned into the SacI site of plasmidpUC118 (J Vieira and J Messing, in Methods in Enzymology, 153 (1987),3-11) in order to generate plasmid pSIV1 which may be converted tosingle stranded DNA for manipulation by site directed metagenesis. ThisDNA region, which includes the SIV env gene (lying between 6090-8298) isthen altered by site directed mutagenesis (I Brierley et al, Cell, 57(1989), 537-547) to introduce a restriction enzyme site for the enzymeEcoRV at positions 6053-6058 using the synthetic oligonucleotide (SEQ IDNO:13)

-   -   5′GAAGAAGGCTATAGCTAATACAT.

A second EcoRV site is then introduced at position 7671-7676 within theSIV env gene corresponding to the cleavage site between the gp120 andgp40 domains of the env gene sequence, using the syntheticoligonucleotide (SEQ ID NO:14)

-   -   5′CAAGAAATAAACTATAGGTCTTTGTGC        to generate the plasmid pSIV2. A DNA fragment (1617 base pairs)        corresponding to the gp120 portion of the SIV env gene is then        prepared by digestion of SIV2 with EcoRV.

The core region of the CMV immediate early gene promoter is obtainedfrom the plasmid pUG-H1 (U A Gompels and A C Minson, 1989, op. cit.) bythe PCR technique using the following two synthetic oligonucleotides(SEQ ID NO:15, SEQ ID NO:16).

upstream primer

-   -   5′ ATC GAATTC CTATAG CCTGGCATTATGCCCAGTACATG        -   EcoRI EcoRV            downstream primer    -   5′TCAAAGCTT CTATAG CCCGGGGAGCTCTGATTATATAGACCTCCC        -   HindIII EcoRV SmaI

The product of this reaction is then cleaved with the enzymes EcoRi andHindIII to generate a DNA fragment which is then cloned into EcoRI- andHindIII-digested plasmid pUC118 to generate the plasmid pCMVIE2 whichhas a unique SmaI site located just downstream of the CMV promotersequence. The EcoRV fragment containing the SIV_(mac) gp120 codingsequence prepared as described above, is then cloned into this SmaIsite, and plasmid pSIV3, with the SIV coding region oriented correctlyto allow expression of the coding sequence from the promoter, is thenselected. This plasmid is then digested with EcoRV to yield ablunt-ended DNA fragment consisting of the SIV sequence together withthe CMV promoter, which is then cloned into PvuII-digested pGH2 (FIG. 2)to produce pGH-120).

D-B. Construction of the SIV gp120 Carrying Recombinant gH− HSV

DNA is purified from the gH− HSV virus constructed as detailed in theprevious section, and co-transfected into gH+ complementing cells alongwith purified pGH-120 DNA. Progeny virus isolated from this transfectionprocedure is then plated on monolayers of the gH+ complementing cellline by standard plaque assay as before using an agar overlay in thepresence of X-gal. The parental gH− virus carries a functionalβ-galactosidase gene, located within the residual gH coding sequences,and in the presence of X-gal, will form blue plaques. Recombinantviruses however, which have acquired the SIV gp120 coding sequence inplace of the β-galactosidase gene, will produce white plaques. Virus isrecovered from these white plaques by picking plugs of agar, and virusstocks prepared through growth of the virus in the gH+ complementingcell line. Virus DNA is prepared from these stocks, and checked for thepresence of the correct DNA structure around the gH gene by SouthernBlotting using appropriate probes derived from the SIV coding sequence.Finally stocks of the virus are prepared as before for vaccinationstudies in animals.

Vaccines comprising the attenuated virus can be prepared and usedaccording to standard techniques known in the art. For example, thevaccine may also comprise one or more excipients and/or adjuvants. Theeffective dose of the attenuated virus to be provided by the vaccine maybe determined according to techniques well known in the art.

Section E. Construction of a gH Defective Recombinant Type 2 HerpesSimplex Virus (DISC HSV-2)

E-A. The HSV2 gH Gene

(a) The Herpes Simplex type 2 (HSV2) gH gene is contained within twoBamH1 restriction fragments of the 25766 strain of HSV2. pTW49 is theBamH1 R fragment of HSV2 strain 25766 cloned into pBR322. pTW54 is theBamH1 S Fragment of HSV2 strain 25766 cloned into pBR322. Theconstruction of a single plasmid containing the complete gH gene isshown in FIG. 23. pTW49 was digested with BamH1 and Sall, and an 870base pair (bp) fragment isolated from an agarose gel. Similarly pTW54was digested with BamH1 and Kpn1 and a 2620 bp fragment isolated from anagarose gel. The two fragments were ligated together with the plasmidpUC119 cut with Sall and Kpn1, resulting in the plasmid pIMMB24.

(b) pIMMB24 was digested with Sall and Kpn1. In addition the plasmid wasdigested with Dra1 (which cuts in the vector sequences), to aid inisolation of the 3490 bp insert. The 3490 bp insert containing the HSV2sequences was purified from an agarose gel. It was then sonicated, theends repaired using T4 DNA polymerase and Klenow, and size fractionatedon an agarose gel. A fraction containing DNA molecules of approximately300-600 bp in length was ligated into M13mp11 cut with Smal (AmershamInternational UK). The ligated mixture was transformed into E. colistrain TG1, and individual plaques were picked. Single-stranded DNA wasmade from each plaque picked, and was sequenced using the dideoxy methodof sequencing, either with Klenow enzyme or with Sequenase, and using³⁵S dATP.

In addition to sequencing in M13 using an oligonucleotide priming fromwithin the M13 sequences, sequence data was also obtained by sequencingdirectly from the pIMMB24 plasmid using oligonucleotide primers designedfrom sequence already obtained. In order to obtain sequence from regionsflanking the gH gene, some sequence information was also obtained fromthe plasmid pTW49.

Because of the high G+C ratio of HSV2 DNA, there were several sequenceinterpretation problems due to ‘compressions’ on the gels. These haveyet to be resolved. In a small number of places therefore, the presentsequence represents the best guess as to what the correct sequence is,based on comparisons with the previously published HSV1 sequence.

(c) The sequence of HSV2 strain 25766 (SEQ ID NO:5) in the region of thegH gene is shown in FIG. 24, along with a translation of the gH insingle letter amino acid code (SEQ ID NO:6). FIG. 25 shows a comparisonof the DNA sequence of HSV1 (SEQ ID NO:7) and HSV2 (SEQ ID NO:5) in thisregion. FIG. 26 shows a comparison of the deduced amino acid sequencesof the HSV1 (SEQ ID NO:8) and HSV2 (SEQ ID NO:6) gH proteins. At the DNAlevel the overall identity is 77%. At the protein level the overallidentity is also 77%, with a further 9.7% of amino acids being similarin properties. The degree of sequence similarity varies to some extentalong the length of the gene, as can be seen from FIG. 27, which showsgraphically the level of similarity. Even more marked than the variationalong the gH gene is the difference in levels of identity between HSV1and HSV2 at the DNA level between the coding and non-coding regions. Ascan be seen from FIG. 25, the nucleotide sequence identity is higherwithin the coding sequence of the gH gene than it is in the intergenicregions. FIG. 27 shows this in a graphical form, with the positions ofthe TK, gH and UL21 genes marked.

(d) The availability of nucleotide sequence data from around the HSV-2gH gene enables further constructs to be made eg it allows the design ofrecombination vectors which enables precise deletion of the gene fromthe viral genome. Because of the differences between HSV1 and HSV2,particularly between the genes, may not have been possible fromknowledge of the HSV1 sequence alone.

Oligonucleotides MB57 (SEQ ID NO:9), MB58 (SEQ ID NO:10), MB59 (SEQ IDNO:11), and MB75 (SEQ ID NO:17) were designed to isolate and clone theregions of sequence flanking the HSV2 gH gene. As shown in FIG. 29, theoligonucleotides were used in a polymerase chain reaction (PCR) toamplify fragments of DNA from either side of the gene. Restriction siteswere included in the oligonucleotides so that the resultant fragmentscontained these sites at their ends, enabled cloning of the fragmentsinto a suitably cut plasmid. The following oligonucleotides, based onthe HSV2 sequence, were used for this purpose:

-   -   Hpal        Inside right MB57 (SEQ ID NO:9) TCAGTTAACGCCTCTGTTCCTTTCCCTTC    -   EcoR1        Outside right MB58 (SEQ ID NO:10) TCAGAATTCGAGCAGCTCCTCATGTTCGAC    -   Hpal        Inside left MB75 (SEQ ID NO:17) TCAGTTAACCGTCGTCCCGGCTGCCAGTC    -   Hind111        Outside left MB59 (SEQ ID NO:11) TCAAAGCTTCTGCAGCGCGGCGGGAGGTGG

The position of these oligonucleotides is also shown on FIG. 25.

On the basis of the description given above in relation to HSV-1 andcommon general knowledge, such a plasmid allows the skilled person toproduce a defective HSV-2 virus lacking precisely the sequences for thegH gene (see below). If these same sequences are cloned into a suitablecell carrying a copy of the gH gene deleted from the HSV-2 genome, this‘complementing cell’ can then support the growth of the defective HSV-2virus by providing the gH protein. Because the sequences have beenchosen so that there is no overlap between the sequences in the cell andthe sequences in the virus, the possibility of the virus acquiring thegene from the cell by recombination is virtually eliminated.

E-B. Complementing Cell Lines

It was found that cells expressing the HSV-1 gH gene (F6 cells, see alsoA Forrester et al, Journal of Virology, 66 (1992), pp 341-348) cansupport the growth of an HSV-2 virus lacking the gH gene. However twonew cell lines were made. CR1 cells use the same promoter and gH gene asF6 cells, but the sequences downstream of the gene are truncated so thatthere is no overlap of sequences between the final DISC virus and thecell line. This is very useful since it means that homologousrecombination cannot occur between the DISC virus and the cell line DNA.In the case of F6 cells and the gH-deleted virus in the Forrester paper,where there is overlap, wild-type gH-plus viruses occur by recombinationat about 1 in 10⁶ viruses. Another cell line, CR2, was also made, whichexpresses the gH gene from the HSV-2 strain 25766. This also supportsthe growth of a DISC HSV-2 and also has no overlapping sequences betweenthe virus and the cell.

Polymerase Chain Reaction (PCR) of Flanking Sequences

Viral DNA is purified from virus by standard methods. Flanking sequencesto either side of the gH gene are amplified by PCR using Vent DNApolymerase (from New England Biolabs) which has a lower error rate thanTaq DNA polymerase (see FIG. 30). The oligonucleotides used for PCRinclude restriction site recognition sequences, as well as the specificviral sequences (see below). Two vectors are made, one for the firststage and one for the second stage of recombination. For both vectorsthe right hand flanking sequences start at the same position to theright of the gH gene. The first stage vector has left hand flankingsequences that, in addition to deleting the HSV-2 gH gene, also deletethe 3′ portion of the viral TK gene. The second stage vector has lefthand flanking sequences which restore the complete TK gene, and extendright up to the 5′ end of the gH gene, as desired in the final virus.

The oligonucleotides used are as follows:

-   -   HindIII        MB97 (SEQ ID NO:18) TCGAAGCTTCAGGGAGTGGCGCAGC    -   Hpal        MB96 (SEQ ID NO:19) TCAGTTAACGGACAGCATGGCCAGGTCAAG    -   Hpal        MB57 (SEQ ID NO:9) TCAGTTAACGCCTCTGTTCCTTTCCCTTC    -   EcoRI        MB58 (SEQ ID NO:10) TCAGAATTCGAGCAGCTCCTCATGTTCGAC        Construction of Vectors

The first stage recombination vector, pIMMB47+:

The two PCR fragments made by oligos MB97 (SEQ ID NO:18)-MB96 (SEQ IDNO:19) and by oligos MB57 (SEQ ID NO:9)-MB58 (SEQ ID NO:10) are digestedwith the restriction enzymes appropriate to the sites that have beenincluded in the PCR oligonucleotides. The MB97-MB96 fragment is digestedwith HindIII and Hpal. The MB57-MB58 fragment is digested with Hpal andEcoRI. These fragments are then ligated into the vector pUC119 which hasbeen digested with HindIII and EcoRI. The resultant plasmid is calledpIMMB45 (see FIG. 30).

To allow for easy detection of the first stage recombinants, the E. colibeta-galactosidase gene, under the control of the Cytomegalovirus (CMV)immediate early promoter is inserted into pIMMB45. The CMV promoter plusbeta-galactosidase gene is excised from a suitable plasmid carrying thepromoter and gene using one or more appropriate restriction enzymes. Ifnecessary, the ends are filled in using the Klenow fragment of DNApolymerase. This is the approach taken by the present applicants.However alternative methodologies will be apparent to those skilled inthe art. For example, the beta-galactosidase gene may be under thecontrol of the SV40 promoter, in which case, the gene and promoter canbe excised from the plasmid pCH110 (Pharmacia PL Biochemicals) usingBamHI and TthlllI, and the ends are filled in using the Klenow fragmentof DNA polymerase (MS Ecob-Prince et al, J Gen Virol, 74 (1993), pp985-994). The fragment is gel-purified. The plasmid pIMMB45 is digestedwith Hpal, phosphatased with Calf Intestinal Alkaline Phosphatase (CIAP)to abolish self ligation, and gel-purified. The gel-purified fragmentsare then ligated together to produce the plasmid pIMMB47+ (see FIG. 31).

The second stage recombination vector, pIMMB46:

The two PCR fragments made by oligos MB94-109 (SEQ ID NO:20) and byoligos MB57 (SEQ ID NO:9)-MB108 (SEQ ID NO:21) are digested with therestriction enzymes appropriate to the sites that have been included inthe PCR oligonucleotides. The MB94-MB109 fragment is digested withHindIII and Hpal. The MB57-MB108 fragment is digested with Hpal andEcoRI. These fragments are then ligated into the vector pUC119 which hasbeen digested with HindIII and EcoRI. The resultant plasmid is calledpIIMB46 (see FIG. 32).

The oligonucleotides used are as follows:

-   -   EcoRI        MB108 (SEQ ID NO:21) TCAGAATTCGTTCCGGGAGCAGGCGTGGA    -   Hpal        MB109 (SEQ ID NO:20)        TCAGTTAACTGCACTAGTTTTAATTAATACGTATGCCGTCCGTCCCGGCTGCCAGTC        E-C. Construction of Recombinant Viruses        a) First Stage.

Virus DNA was made from strain HG52-D, which is a plaque-purifiedisolate of the HSV-2 strain HG52. Virus DNA (2.5 μg) and pIMMB47+plasmid DNA (0.25 μg) was transfected into CR1 cells using the CaPO₄precipitation method (Chen & Okayama, Molecular and Cellular Biology, 7,p. 2745). Recombination takes place within the cells, and a mixture ofrecombinant and wild type virus is produced. The mixture wasplaque-purified three times on CR1 cells in the presence of acyclovir(10 μg/ml), to select for TK-minus virus. A single plaque was then grownup and analysed. The virus was titrated on normal Vero cells and on CR1cells. If the virus is a gH-deleted virus, it should only grow on CR1cells and not on Vero cells. Table E-1 shows that this is the case. Itcan be seen that the virus does not grow at all on the non-complementingVero cells even at the highest virus concentrations, but does grow wellon the CR1 complementing cell line, which expresses the HSV-1 gH gene.The virus also grows well on CR2 cells which express the HSV-2 gH gene(data not shown).

TABLE E-1 Growth of first stage recombinant virus on complementing (CR1)and non-complementing (Vero) cells. CR1 (gH+) Vero Virus dilutions 10⁻⁴10⁻⁵ 10⁻⁶ 10⁻¹ 10⁻² 10⁻³ 10⁻⁴ Numbers of >350 174 22 0 0 0 0plaques >350 169 19 0 0 0 0b) Second Stage.

DNA was made from this TK-minus DISC virus and a recombination wascarried out as above with the plasmid pIMMB46. In this case TK-plusrecombinants were selected, on a gH-expressing TK-minus BHK cell line,by growth in medium containing methotrexate, thymidine, glycine,adenosine and guanosine. Virus was harvested and grown again underselective conditions twice more before a final plaque purification wascarried out on CR1. Virus was grown up and analysed by Southernblotting. Virus DNA from the original HG52-D, the TK-minus DISC virus,and the TK-plus DISC virus were digested with BamHI and separated on anagarose gel. The DNA bands were then transferred to nylon membrane bythe Southern blotting method, and probed with radiolabelled fragmentsfrom the right hand flanking sequences. FIG. 33 shows the structures ofthese viruses, with the expected band sizes after BamHI digestion. Theprobe used is marked as ‘R’ beneath a dashed line. The probe shouldhybridise to a different size band in each of these viruses, as follows:

Band size hybridising Virus (base pairs) HG52-D 3481 TK-minus “firststage” DISC virus 3140 TK-plus “second stage” DISC virus 4225

FIG. 34 shows that this is the case. Lane 5 shows the HG52-D virus, Lane2 contains the TK-minus “first stage” DISC virus, and lanes 3, 4, 6, 7and 8 contain TK-plus “second stage” DISC viruses. This confirms thatthe DNA structure in each of these viruses is as expected.

The defective HSV-2 can be used as a vaccine. After growth in thecomplementing cell line, the HSV-2 virus is phenotypically identical toa wild type HSV-2 virus, and can infect cells in a normal manner. Innormal cells the defective HSV-2 virus undergoes a single cycle ofreplication. Viral particles are produced, but because normal cells donot express the gH gene, these HSV-2 viral particles lack the gHprotein, and are subsequently unable to infect further cells.

Furthermore the sequence of the HSV-2 gH gene as hereby provided can bevery useful for several purposes. In the context of the DISC virussystem, it can be useful in that the detailed knowledge of the sequenceallows the making of precise deletions around the gH gene. In addition,should it prove desirable to use the HSV-2 gH gene in the complementingcell line, either to provide better complementation of the gH-deletedvirus, or to produce a type 2 DISC virus with improved immunogenicity,then again the sequences will prove invaluable. Uses in different areascan also be envisaged, such as subunit vaccines using the HSV-2 gHprotein as an immunogen, or recombinant viruses (either HSV or otherviruses) expressing the HSV-2 gH gene. For diagnostic purposes PCRprimers can be designed using the HSV-2 gH sequence, in order, ifdesired, to distinguish between HSV-1 and HSV-2.

Section F. In Vivo Mouse Studies

Protection Studies

The in vivo mouse ear model was used to study prophylactic effects.Equivalent doses of inactivated wild-type (‘w.t.’) HSV-1 (strain SC16,see T J Hill et al, J Gen Virol. 28 (1975), pp 341-353) and DISC HSV-1were compared for their effect on the replication of w.t. HSV-1, theirability to provide protection against w.t. HSV-1 challenge and to induceHSV-specific neutralising antibodies.

4-5 week old BALB/c mice were vaccinated with varying doses of DISCHSV-1 or inactivated virus by scarification in the left ear pinna. Viruswas inactivated using β-propiolactone (see description above in relationto HSV-1). The mice were challenged with 2×10⁶ pfu w.t. HSV-1 (strainSC16) in the opposite ear two weeks after vaccination. The amount ofvirus present in that ear 5 days post challenge was assayed by plaquingon BHK cells. (See FIG. 8.)

It can be seen from FIG. 8 that vaccination with 5×10⁵ and 5×10⁶ pfuDISC HSV-1 (pfu measured on complementing cell line for DISC viruses)led to complete protection against replication of the challenge virus,whilst mice vaccinated with inactivated virus still had live challengevirus present.

A similar result was obtained when virus titres were assayed from theganglia of vaccinated animals 5 days after challenge (data not shown).

Serological Response to Disc HSV-1 Vaccination

The role of antibody in protection conferred by the DISC HSV-1vaccination was investigated. Both neutralising antibody titres andtotal antibody titres, as determined by ELISA, were measured.

Groups of 6 mice were vaccinated with 5×10⁵ pfu of DISC HSV-1, killedDISC HSV-1, w.t. HSV-1 (strain SC16) or with PBS and serum samples takenat 2 and 14 weeks post vaccination. Neutralising antibodies weremeasured in the presence of complement and expressed as the inverse ofthe serum dilution which reduced the number of plaques by 50%. ELISAantibody titres were measured on plates coated with HSV-1 infected BHKcell lysates and titrated to endpoint. (See FIG. 9.)

It can be seen from FIG. 9 that no significant differences in antibodytitres were observed between animals vaccinated with DISC HSV-1 and anequivalent amount of killed DISC HSV-1.

Delayed-Type Hypersensitivity (DTH) Response to Disc HSV-1 Vaccination

The importance of a DTH response in protection against herpes virusinfection has been well documented. The ability of, the DISC HSV-1 toraise a DTH response was investigated by vaccinating groups of mice withDISC HSV-1, killed DISC HSV-1, and live w.t. HSV-1, by scarification ofthe left ear pinna. Four doses (5×10³, 5×10⁴, 5×10⁵ and 5×10⁶ pfu) ofvaccine were used, and two weeks later the vaccinated animals werechallenged in the opposite ear with 10⁶ pfu w.t. HSV-1 (strain SC16).The DTH response at the site of challenge was assessed by measurement ofear thickness at 24 and 48 hours post challenge and expressed as thedifference between the challenged and unchallenged ears. (See FIG. 10.)

It can be seen from FIG. 10 that at low vaccine doses (5×10³, 5×10⁴pfu), no DTH response was observed with killed DISC HSV-1, whilst aclear DTH response was demonstrated after DISC HSV-1 vaccination. Athigh doses (eg 5×10⁶ pfu), both the DISC HSV-1 vaccine and killed DISCHSV-1 preparations induced similar DTH responses.

The DTH responses induced by different doses of the various vaccinepreparations thus correlate with their protective effect againstchallenge virus replication. The efficacy of vaccination with low dosesof the DISC HSV-1 vaccine may therefore be due to the induction of Tcell-mediated immunity.

Demonstration that Disc HSV Type 1 Virus is Capable of GeneratingCytotoxic T Cells

Cytotoxic T cells have been shown to be involved in the protectionagainst, and recovery from, primary HSV infection. DISC HSV-1 vaccinatedmice were therefore studied for the presence of HSV-1 specific cytotoxicT cell activity.

Cytotoxic T cell activity following immunisation was generated andassayed according to standard procedures eg as exemplified in S Martinet al, J Virol, 62 (1988), 2265-2273, and W S Gallichan et al, J InfectDis, 168 (1993), 622-629. More specifically, groups of female BALB/cmice were immunised intra-peritoneally with 2×10⁷ pfu of virus (DISCHSV-1; killed DISC HSV-1; MDK a thymidine kinase negative HSV-1 strain)on day 0 and the immunisations repeated (same dose and route) after 3weeks. A group of control mice received 0.1 ml of PBS intraperitoneallyat the same time points. Ten days after the second immunisation thespleens of the mice were removed and pooled for each group.

Spleens were also removed from unimmunised BALB/c mice for thepreparation of feeder cells (16 feeder spleens being sufficient for 4groups of six effector spleens). All subsequent steps were performed ina laminar flow hood using aseptic technique. The spleens were passedthrough a sterile tea-strainer to produce a single cell suspension inRPMI 1640 medium supplemented with 10% heat inactivated foetal calfserum (effector medium). Debris was allowed to settle and the singlecell suspension was transferred to a fresh container. The cellsuspensions were washed twice in effector medium (1100 rpm, 10 minutes)and then passed through sterile gauze to remove all clumps. The effectorspleen cell suspensions were then stored on ice until required.

Feeder spleen cells were resuspended to 1×10⁷ cells/ml in effectormedium and mitomycin C was added to a final concentration of 20 μg/ml.The feeder cells were incubated at 37° C. for 1 hour. Feeder cells werewashed four-times in PBS supplemented with 1% FCS and once in PBS withno protein. Live virus (MDK) was added to the mitomycin C treated feedercell pellet at a concentration of 3 pfu of virus per spleen cell.Following a one hour incubation at 37° C. the feeder cells were washedonce with effector cell medium.

Effector cells were resuspended to 5×10⁶ cells/ml, whilst feeder cellswere resuspended to 2.5×10⁶ cells/ml. 500 μl of effector cell suspensionand 500 μl feeder cell suspension were added to the wells of a 24 wellplate. The plates were incubated in a humid atmosphere at 37° C. (5%CO₂) for 4 days.

The effector and feeder cells were harvested from the 24 well plate. Thecells were spun down once and the pellet resuspended in effector medium(5 ml of medium per 2 plates). The cell suspension was layered ontolymphocyte separation medium and spun at 2500 rpm for 20 minutes. Thelive effector cells were harvested from the interface and washed twice,once at 1500 rpm for 15 minutes and once at 1100 rpm for 10 minutes. Theeffector cells were finally resuspended at the required concentration ineffector medium and stored on ice until required.

Labelled target cells were prepared for the cytotoxicity assay.Uninfected syngeneic A202J target cells A20/2J cells were harvested fromtissue culture flasks: 2×10⁷ cells were added to each of 2 containers(to become infected and uninfected targets). The cells were washed withDMEM (with no additions). To the infected cells live MDK virus was addedat 10 pfu per cell and an equivalent volume of EMEM was added to theuninfected cells. One mCi of 51Cr was added to each of the universalsand the cells were incubated at 37° C. (in a waterbath) for 1 hour. Thetarget cells were then washed three times (10 minutes, 1100 rpm) intarget medium (DMEM supplemented with 10% FCS) and finally resuspendedto the required cell concentration in target cell medium. Bothuninfected and infected target cells were resuspended to 1×10⁶ cells/mland 1×10⁵ cells/ml and 100 μl (ie to give 1×10⁵ targets/well and 1×10⁴targets/well respectively) was plated out into the appropriate wells ofa round bottomed 96 well plate. All experimental points were set up inquadruplicate. Each effector cell type was resuspended to 8×10⁶ cells/mlin effector medium and two-fold dilutions were prepared. 100 μl of theeffector cell suspensions were added to the wells containing thelabelled target cells to give 8×10⁵ effector cells/well, 4×10⁵ effectorcells/well, 2×10⁵ effector cells/well and 1×10⁵ effector cells/well.Thus with 10⁵ target cells per well, effector to target ratios were:8:1, 4:1, 2:1 and 1:1. With 10⁴ target cells per well the effector totarget ratios were 80:1, 40:1, 20:1 and 10:1. Maximum chromium releasefor each target cell type was obtained by adding 100 μl of 20% TritonX-100 to wells containing target cells only (ie no effectors). Thespontaneous release for each target cell type was obtained by theaddition of 100 μl effector cell medium to wells containing target cellsonly.

The plates were incubated at 37° C. for four hours in a humidatmosphere. After this time the plates were spun for four minutes at1500 rpm and 100 μl of supernatant was removed from each of the wells.The supernatant was transferred to LP2 tubes and radioactivity containedin the tubes was then counted for 1 minute on a gamma counter. The %specific chromium release was determined using the formula

$\mspace{56mu}{{\%\mspace{14mu}{specific}\mspace{14mu}{release}} = {\frac{{{{Exp}.\mspace{14mu}{mean}}\mspace{14mu}{cpm}} - {{{spon}.\mspace{14mu}{mean}}\mspace{14mu}{cpm}}}{{{{Max}.\mspace{14mu}{mean}}\mspace{14mu}{cpm}} - {{{spon}.\mspace{14mu}{mean}}\mspace{14mu}{cpm}}} \times 100}}$$\begin{matrix}{{{Exp}.} = {Experimental}} \\{{{Spon}.} = {Spontaneous}} \\{{{Max}.} = {Maximum}}\end{matrix}$

The results are shown in FIG. 11 and Table F-1

TABLE F-1 Inactivated E:T ratio DISC HSV-1 Virus MDK Unvaccinated 8:153.9 1.5 48.3 ND 4:1 49.6 0.0 42.2 0.0 2:1 36.9 0.0 31.0 0.0 1:1 23.90.0 21.9 0.0 % HSV-1 Specific Lysis (% lysis of HSV-infected cells minus% lysis of uninfected cells).

DISC HSV-1 vaccination induced HSV-1 specific CTL activity comparable tothat produced by infection with the fully replicative MDK virus. Incontrast no HSV-1 specific CTL activity was observed in mice immunisedwith killed DISC HSV-1 or in PBS treated animals, although somenon-specific killing was observed in these animals. The reason for thisis not clear, but it could represent a high level of NK cell activity.

Vaccination of mice with the DISC HSV-1 has thus been shown to induceantibody, CTL and DTH activity against HSV-1 virus antigens. The abilityto activate both humoral and cell-mediated immune responses against abroad spectrum of virus proteins may explain the effectiveness of theDISC virus vaccination.

Long-Term Protection

The in vivo mouse ear model was used to study long term prophylacticeffect of DISC HSV-1

4-5 week old BALB/c mice were divided into groups containing 6 animalseach.

The groups were vaccinated as follows:

Group Vaccination PBS Mock immunisation with PBS 1K 1 immunisation withinactivated DISC HSV-1 2K 2 immunisations with inactivated DISC HSV-1 1L1 immunisation with (live) DISC HSV-1 2L 2 immunisations with (live)DISC HSV-1 1S 1 immunisation with w.t. HSV-1 (strain SC16) 2S 2immunisations with w.t. HSV-1 (strain SC16)

All groups were immunised by scarification of the left ear pinna with5×10⁵ pfu on day 0 and blood samples taken on days 15, 27, 90, 152 and218. Groups PBS, 2K, 2L and 2S received additional immunisations of PBSor 5×10⁵ pfu on day 20. All groups were challenged with 5×10⁵ w.t. HSV-1(strain SC16) on day 223. The amount of virus present in the challengedear (right) 5 days post challenge was assayed by plaquing on BHK cells.The results as depicted by FIG. 20 show that two vaccinations with DISCHSV-1 (group 2L) provides goods protection compared to inactivated DISCHSV-1 (group 2K), but that better protection was obtained with w.t.HSV-1 (strain SC16). The efficacy of vaccination with w.t. HSV-1 is ofcourse, to be expected. However the use of normal live viruses asvaccines is generally undesirable. FIG. 21 shows the neutralisingantibody titres induced by the various vaccinations. This shows thatsince 2 doses of DISC HSV-1 produce the same titre as two doses of theinactivated DISC HSV-1, the protective effect of DISC HSV-1 cannot besimply explained by antibody induction.

Prophylactic Effect of Disc HSV-2

The in vivo mouse ear model was used to study the prophylactic effect ofDISC HSV-2.

Six week old BALB/c mice were divided into groups. They were immunisedby scarification of the left ear pinna as follows.

Group Vaccination Material and Dose 1 5 × 10² pfu live DISC HSV-2 2 5 ×10³ pfu live DISC HSV-2 3 5 × 10⁴ pfu live DISC HSV-2 4 5 × 10⁵ pfu liveDISC HSV-2 5 5 × 10² pfu killed DISC HSV-2 6 5 × 10³ pfu killed DISCHSV-2 7 5 × 10⁴ pfu killed DISC HSV-2 8 5 × 10⁵ pfu killed DISC HSV-2 95 × 10⁴ pfu w.t. HSV-2 (strain HG52) 10 5 × 10⁵ pfu w.t. HSV-2 (strainHG52) 11 PBS (The DISC HSV-2 was a gH deletion mutant of strain HG52.)

Three weeks later, all groups were challenged by scarification of theright ear pinna with 5×10⁴ of w.t. HSV-2 (strain HG52).

The amount of virus present in the challenged ear (right) 5 days postchallenge was assayed by plaquing on BHK cells (see FIG. 22). Theresults as depicted by the figure show that vaccination with DISC HSV-2at doses of 5×10³, 5×10⁴ and 5×10⁵ pfu provides good protection againstchallenge with w.t. HSV-2 (strain HG52) compared to killed DISC HSV-2.However and as is to be expected, better protection was obtained withw.t. HSV-2 at doses of 5×10⁴ and 5×10⁵ pfu, but the use of normal livewild type viruses as vaccines is undesirable.

Section G. In Vivo Guinea Pig Studies

As mentioned earlier, HSV-2 appears to be closely associated withgenital lesions. The guinea pig currently provides the best animal modelfor primary and recurrent genital disease in humans (L R Stanberry etal, J Infect Dis 146 (1982), pp 397-404).

Therefore the applicants have extended the above-described mouse studiesto the guinea pig vaginal model of HSV-2 infection which provides auseful system to assess the immunogenicity of candidate vaccines againstgenital HSV-2 infection in humans. It permits a comprehensive assessmentof primary clinical symptoms following intra-vaginal challenge withHSV-2, and also analysis of the frequency of subsequent recurrences.

(1) Groups of 14 animals were immunised with two doses of the DISC HSV-1vaccine (2×10⁷ pfu, 3 weeks apart) either by non-traumatic introductioninto the vagina (intra-vaginal route), or by scarification of the earpinna (intra-epithelial route). A control group of 21 animals wasvaccinated intra-vaginally with a mock virus preparation and a furthergroup of 14 animals was vaccinated intra-epithelially with twoequivalent doses of β-propiolactone-inactivated w.t. HSV-1.

Vaccinated animals were challenged 3 weeks later with 10^(5.2) pfu w.t.HSV-2 virus (strain MS) and monitored for the symptoms of primary andrecurrent disease.

(a) Following w.t. HSV-2 challenge, animals were assessed daily over atwo week period for symptoms of primary infection. Clinical lesions werescored as a direct numerical value, and erythema was scored on a scaleof 1-5. The vaginal area was also measured as an index of oedema (datanot shown). The results are shown in FIGS. 12 and 13. Points on thegraphs represent mean erythema score per animal per day (FIG. 12) andmean total lesion score per day per animal (FIG. 13).

The results show that intra-epithelial and intra-vaginal vaccinationwith the DISC HSV-1 both provided a high degree of protection againstthe primary symptoms of HSV-2 infection. Surprisingly, inactivated HSV-1administered by the intra-epithelial route also provided substantialprotection, though apparently less than that afforded by the DISC virusvaccine.

(b) Daily vaginal swabs were taken from all animals over a 12 day periodpost-challenge and virus titres determined by plaquing on Vero cells inorder to monitor growth of the challenge virus in the vagina. Theresults as depicted in FIG. 14 shows that infection virus titres inmock-vaccinated animals rose to a maximum of 3×10⁴ at day 2 postchallenge, and could be detected until day 10. By contrast, virus titresin the vaccinated animals declined steadily from day 1, and wereundetectable by day 7. No significant different was observed between thegroups immunised with the DISC HSV-1 or the inactivated viruspreparation.

(c) Following HSV-2 challenge, animals which had fully recovered fromthe acute phase of disease by 28 days were monitored daily for a further100 days for the recurrence of disease. Numbers of animals in each groupwere: DISC/Intra-vaginal—14; DISC/Intra-epithelial—12:Inactivated/Intra-epithelial—14; Mock/Intra-vaginal—12. Clinical lesionswere scored as a direct numerical value, and erythema was scored on ascale of 1-5. The results are shown in FIGS. 15 a and 15 b. Points onthe graphs represent the cumulative totals of mean values per day peranimal.

The results show that animals vaccinated with the DISC HSV-1 by theintra-vaginal route showed approximately a 50% reduction in the numberof recurrent HSV-2 lesions occurring over the 100 day follow-up period.Intra-epithelial vaccination with DISC HSV-1 and inactivated virus alsoresulted in a reduction of recurrent lesions, but to a lesser extent.

(2) The following experiment was also designed to assess theimmunogenicity of candidate DISC vaccines based on HSV-1 against genitalHSV-2 infection. The experiment was designed to compare differentvaccination routes (per vaginum, oral and nasal ie different mucosalsurfaces) and different doses of either DISC HSV-1 or inactivated HSV-1in the guinea pig.

Materials and Methods

Virus:

(i) DISC HSV-1 was propagated on Vero cells (F6) which had beentransfected with the HSV-1 gH gene as described above. Briefly,confluent monolayers of F6 cells were infected with DISC HSV-1 at amultiplicity of 0.1 pfu per cell and harvested when 90-100% cpe wasobserved. Cells were harvested with a cell scraper, pelleted bycentrifugation and the pellet resuspended in a small volume of EaglesMinimum Essential Medium (EMEM). The suspension was sonicated for 1minute and stored in aliquots at −70° C. Virus titres were determined onF6 cells.

(ii) DISC HSV-1 was inactivated by the addition of β-propiolactone at aconcentration of 0.05% for one hour at room temperature. Inactivationwas checked by adding the virus to F6 cells.

(iii) HSV-2 strain MS was propagated and titred on Vero cells in thesame manner as DISC HSV-1 as described above.

Animals: Female Dunkin-Hartley guinea-pigs (300-350 g) were obtainedfrom Davis Hall, Darley Oaks Farms, Newchurch, Nr. Burton-on-Trent.

Experimental design: Groups of 12 animals were immunised with two dosesof 8×10⁶ pfu DISC HSV-1 or with equivalent doses of inactivated DISCHSV-1, on days 1 and 17 of the experiment. Immunisation was performedwith either 0.05 ml of virus intravaginally, with 0.2 ml of virusintranasally or with 0.2 ml virus orally. A control group of 12 animalswas vaccinated intravaginally with a mock preparation of virusconsisting of sonicated Vero cells. All groups were challengedintravaginally on day 34 with 10^(5.2) pfu HSV-2 (strain MS) and theexperiment blinded by randomisation of the cages by an independentworker. For a period of 11 days following challenge, animals weremonitored for the symptoms of primary disease. Clinical observationswere scored as the number of lesions present in the vaginal area and thepresence of erythema (scored on a scale of 1-5). In addition, dailyvaginal swabs were taken from all animals over a 12 day period postchallenge and virus titres were determined by plaquing on Vero cells inorder to monitor growth of the challenge virus in the vagina.

Statistical methods: Differences in group clinical scores were testedfor significance using the Mann-Whitney U test. Values of p<0.1 wereconsidered significant.

Results:

Clinical disease profile. The mean lesion score per animal, the meanerythema score and the effect of vaccination on post challenge virusreplication for each of the immunisation groups are shown in FIGS. 16,17 and 18 respectively. As compared to mock vaccinated animals,vaccination with DISC HSV-1 by the intravaginal route provided a highdegree of protection from primary symptoms of infection. In contrast,vaccination with inactivated DISC HSV-1 at an equivalent dose did notlead to any significant protection.

Intranasal immunisation with DISC HSV-1 resulted in an even higherdegree of protection than intravaginal vaccination. This wasparticularly apparent when looking at the number of days with severedisease, as defined by a lesion score of 6 or more (see table G-1).Inactivated DISC HSV-1 gave some protection via the intranasal route,but it was not as effective as vaccination with DISC HSV-1.

Vaccination via the oral route also led to protection, but to a lesserdegree than intranasal or intravaginal vaccination. Again vaccinationwith DISC HSV-1 virus protected more efficiently than vaccination withinactivated DISC HSV-1.

TABLE G-1 INCIDENCE OF PRIMARY DISEASE SYMPTOMS Any dis- Disease on-ease symp- Lesion score Duration of going on day Immunisation toms (% >5(% of disease (mean 11 (% of with of animals) animals) no. days)animals) mock 92 75 6.8 75 DISC HSV-1 33 17 4.5 8 i.vag HSV-1 92 67 6.283 inactivated i.vag DISC HSV-1 33 0 2.3 0 i.nas HSV-1 67 17 6.3 42inactivated i.nas DISC HSV-1 90 20 4.1 20 oral HSV-1 91 36 5.8 64inactivated oral

Thus the following conclusions can be drawn from this experiment withthe in vivo guinea pig model.

A. Vaccination with DISC HSV-1 via the intravaginal and intranasalroutes led to a high degree of protection from acute disease symptomsfollowing a challenge with HSV-2.

B. Intranasal administration of DISC HSV-1 gave the highest degree ofprotection when considering the number of days of severe disease (asdefined by the presence of 6 or more lesions).

C. Intravaginal vaccination with inactivated virus resulted in clinicaldisease symptoms similar to those observed in mock-infected guinea-pigs.Intranasal vaccination with inactivated DISC HSV-1 gave a significantdegree of protection, but not as high as DISC HSV-1 vaccination via thisroute.

D. A significant difference was observed between disease symptoms inanimals vaccinated orally with DISC HSV-1 and mock-infected animals.However, this degree of protection was less than that observed inanimals vaccinated with DISC HSV-1 via the intranasal or intravaginalroute.

E. Symptoms in animals vaccinated orally with inactivated DISC HSV-1were not significantly different from those in the mock-infected group.

F. The data on shed virus is interesting. Surprisingly the per vaginumvaccination route resulted in significantly lower levels of recoveredvirus following the challenge dose. This may be due to local antibodyproduction.

(3) The following experiment was designed to investigate HSV-2 inducedrecurrent disease following therapeutic vaccination.

This was of interest as it has previously been shown that therapeuticadministration of certain recombinant HSV-2 antigens, together withadjuvant, can decrease the frequency of subsequent recurrences (see L RStanberry et al, J Infect Dis 157 (1988), pp 156-163; L R Stanberry etal, J Gen Virol, 70 (1989) pp 3177-3185; and R J Y Ho et al, J Virol, 63(1989), pp 2951-2958).

Accordingly 21 animals which had recovered fully from primary HSV-2disease four weeks after challenge were randomised into three groups,and treated with live DISC HSV-1 intravaginally (10 animals), orintra-epithelially (11 animals). A group of 12 animals, which hadpreviously acted as controls for prophylactic vaccination (see (2)above) and which had also recovered fully from primary disease weretreated with an equivalent mock preparation (12 animals). The animalswere given further identical treatments 24 and 48 days later. Thefrequency of recurrent disease was monitored from the day of firsttreatment for a further 100 days, and the cumulative results are shownin FIG. 19 and summarised in Table G-2 below.

TABLE G-2 Effect of therapeutic vaccination on recurrent disease DISCHSV-1 DISC HSV-1 Mock Intra-epithelial Intra-vaginal Total % of MockTotal % of Mock Total % of Mock 1 Mean total disease/days 9.41 100 6.9073 7.32 78 per animal 2 Mean total episodes per 6.27 100 4.67 74 5.10 81animal 3 Disease incidence 12/12 100 9/11 82 10/10 100 4 Severity perepisode 3.21 100 3.00 93 2.86 89 Mean duration of 1.49 1.27 1.38 episode(days) 1 Total number of days where disease was observed (either lesionsor erythema) over the whole observation period (100 days from 1 monthafter challenge with HSV-2) 2 Total of days disease episodes over thewhole observation period (episode length defined as period between twoconsecutive disease-free days 3 Proportion of animals showing any lesionor erythema score during whole observation period 4 Total sum oferythema scores and lesion numbers over the whole observation perioddivided by number of episodes observed

It can be seen that each of the groups treated with DISC HSV-1 appearedto experience a modest reduction (about 25%) in the overall number ofdisease/days and episodes especially over the 50 day period followingsecond vaccination.

Sera were collected from these animals at the end of the 100 dayobservation period. The ELISA and NT antibody titres in the sera werenot significantly higher than those recorded post-challenge but beforetherapeutic treatment and there were no significant differences intitres between the mock-treatment group and the DISC HSV-1 treatedgroups. Thus therapeutic administration of DISC HSV-1 virus eitherintra-vaginally or intra-epithelially resulted in an apparent reduction(20-25%) in the frequency of recurrence compared with mock-treatedanimals.

(4) The following experiment was designed to investigate the therapeuticvalue of a DISC virus based on HSV-2. A DISC HSV-2 (strain HG 52) havinga deletion of the gH gene was made as described earlier and inaccordance with the general teaching hereof, also using standardprocedures in the art. The DISC version of the strain was grown in Verocells transfected with the HSV-2 gH gene also in accordance with theteaching hereof.

The experiment was a head to head comparison of DISC HSV-1 with DISCHSV-2 in female 350-400 gms guinea-pigs. Guinea-pigs were divided intothree groups. All guinea-pigs were infected with 10^(5.8) pfu HSV-2strain MS. Four weeks were then allowed for the primary disease to haveboth developed and resolved and for recurrences to have started. Theanimals were then treated. A first group of 15 animals was treatedintravaginally with a mock preparation of virus consisting of sonicatedVero cells. A second group of 13 animals was treated intravaginally with10⁷ pfu DISC HSV-1. A third group of 14 animals was treatedintravaginally with 10⁷ pfu DISC HSV-2. Treatment was repeated in 14days. The results are shown in Table G-3. Days 1-13 covers the periodbetween the two treatments. Days 14-27 covers the two week periodsubsequent to the second treatment. Days 1-27 covers the completeperiod.

As shown by the results, it appears that treatment with DISC HSV-2 waseffective in alleviating symptoms caused by infection with HSV-2 strainMS. Treatment with DISC HSV-2 was more effective than treatment withDISC HSV-1.

The invention described and the disclosure made herein are susceptibleof many modifications and variations as will be apparent to, and readilyperformable by, the skilled reader: and the disclosure extends tocombinations and subcombinations of the features mentioned and/ordescribed herein. Documents cited herein are hereby incorporated byreference.

TABLE G-3 Erythema scores Lesions scores Disease Days Group Total Peranimal % of Mock Total Per animal % of Mock Total Per animal % of MockDays 1-13 Mock 38 2.53 100 66 4.40 100 42 2.80 100 DISC HSV-1 34 2.62103 48 3.69 84 34 2.62 93 DISC HSV-2 22 1.57 62 40 2.86 65 26 1.86 66Days 14-27 Mock 13 0.87 100 23 1.53 100 17 1.13 100 DISC HSV-1 9 0.69 8014 1.08 70 11 0.85 75 DISC HSV-2 2 0.14 16 3 0.21 14 3 0.21 19 Days 1-27Mock 51 3.40 100 89 5.93 100 59 3.93 100 DISC HSV-1 43 3.31 97 62 4.7780 45 3.46 88 DISC HSV-2 24 1.71 50 43 3.07 52 29 2.07 53

1. A vaccine comprising a pharmaceutically acceptable excipient and aneffective immunizing amount of a mutant virus, wherein said mutant virusis a mutant poxvirus and has a genome which has an inactivating mutationin a viral gene, said viral gene being essential for the production ofinfectious new virus particles, wherein said mutant virus is able tocause production of infectious new virus particles in a complementinghost cell expressing a gene which complements said essential viral gene,but is unable to cause production of infectious new virus particles whensaid mutant virus infects a host cell other than a complementing hostcell; for prophylactic or therapeutic use in generating an immuneresponse in a subject.
 2. The vaccine of claim 1 wherein the poxvirus isan orthopoxvirus.
 3. The vaccine of claim 2 wherein the poxvirus is avaccinia virus.